Treating Vascular Events with Statins by Inhibiting PAR-1 and PAR-4

ABSTRACT

The present invention relates to new methods for treating and/or preventing vascular events by inhibiting G-coupled Protease Activating Receptor (PAR)-1 and/or PAR-4 with the administration of statins. In one embodiment, individuals who are at risk for vascular events, but have cholesterol levels (e.g., total cholesterol or Low Density Lipoprotein) in normal ranges, are treated with statins.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/811,563, filed on Mar. 29, 2004, entitled “Treating Vascular Eventswith Statins by Inhibiting PAR-1 and PAR-4” by Victor L. Serebruany.

The entire teachings of the above application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

Coronary Heart Disease (CHD) is one of the most common diagnoses ofhospital patients in the United States, with over five million casesoccurring yearly. High levels of bad cholesterol (Low DensityLipoprotein (LDL-C)), total cholesterol (Total-C) or triglycerides areoften associated with increased incidence of CHD, stroke and othervascular events. These patients are frequently treated with lipidlowering drugs, such as statins. In certain aspects, the biologicalmechanisms of these statins remain unclear.

However, many individuals exist who have normal levels of badcholesterol, total cholesterol, or triglycerides, but still suffer fromor are at risk for such vascular events. These vascular events poseserious problems to patients and the physicians who treat them.Physicians continue to search for better preventative and/or curativetreatments for vascular diseases.

Hence, a need exists for new and improved treatment options forindividuals who have vascular diseases, particularly for thoseindividuals having cholesterol levels in normal ranges. Additionally, aneed exists to determine the mechanisms underlying vascular disorders,so that better therapies which target these patient types can bedeveloped.

SUMMARY OF THE INVENTION

The present invention is based on the surprising discovery that statins,a cholesterol lowering drug, inhibit a G-coupled Protease ActivatingReceptor (PAR)-1 and/or PAR-4 (e.g., platelet PAR-1 and/or plateletPAR-4). This newly discovered mechanism has led to new methods fortreating individuals who have a vascular disease or who are at risk fordeveloping such a disease.

Accordingly, the present invention relates to methods for treating avascular disorder in an individual by assessing the levels of PAR-1,PAR-4, or both. When the individual has an elevated level of PAR-1,PAR-4 or both, then the method includes administering to the individualan effective amount of a statin to reduce the levels of PAR-1, PAR-4 orboth.

The present invention also embodies methods for reducing a PAR-1 level,a PAR-4 level, or both in an individual, by selecting an individualhaving an elevated PAR-1 level, an elevated PAR-4 level, or both; andadministering to the individual an effective amount of a statin toreduce the PAR-1 level, the PAR-4 level or both. In one embodiment, thepresent invention pertains to selecting individuals that also havetotal-C levels, LDL-C levels or triglyceride levels that are notconsidered to be high (e.g., in the normal or low ranges). Morespecifically, the present invention includes individuals with a Total-Clevel of less than about 200 mg/dL (e.g, normal range is considered tobe between about 60 mg/dL and about 199 mg/dL). Similarly, the presentinvention further embodies individuals having LDL-C levels that are lessthan about 130 mg/dL (e.g., normal range is between about 85 mg/dL andabout 129 mg/dL). Yet, the present invention can also be practiced onindividuals having triglyceride levels of less than 150 mg/dL (e.g., anormal range is between about 30 mg/dL and about 149 mg/dL).

As described herein, PAR-1, PAR-4 or both can be reduced by at leastabout 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), as comparedto a control. Examples of statins include, but are not limited to,atorvastatin, pravastatin, fluvastatin, cerivastatin, lovastatin,simvastatin, rosuvastatin, pitavastatin, and metabolites thereof.Effective amounts of statins include oral administration in an amountbetween about 5 mg and about 250 mg per day, as further describedherein.

The present invention, in another embodiment, relates to methods fortreating an individual with a vascular disorder or preventing a vasculardisorder in an individual who is as risk for developing one. This methodis performed by selecting an individual that has a vascular disorder;and a Total-C level of less than 200 mg/dL, or a LDL-C level of lessthan 130 mg/dL, or both; and administering to the individual aneffective amount of a statin to inhibit PAR-1, PAR-4 or both. Vasculardisorders include, but are not limited to, myocardial infarction,angina, stroke, pulmonary embolism, transient ischemic attack, deep veinthrombosis, thrombotic re-occlusion subsequent to a coronaryintervention procedure, heart surgery or vascular surgery, peripheralvascular thrombosis, Syndrome X, heart failure and a disorder in which anarrowing of at least one coronary artery occurs. Statins areadministered, and PAR-1 and/or PAR-4 are reduced, as further describedherein.

When statins inhibit PAR-1 and/or PAR-4, statins, in turn, reduce orprevent thrombin generation. Consequently, the present inventionincludes methods for reducing or preventing thrombin generation in anindividual. The method involves selecting an individual that haselevated PAR-1, PAR-4 or both; or selecting an individual that has avascular disorder; and a Total-C level of less than 200 mg/dL, or aLDL-C level of less than 130 mg/dL, or both. After the individual isselected, the method includes administering to the individual aneffective amount of a statin to inhibit PAR-1, PAR-4 or both. Thesesteps reduce the amount of thrombin generation, as compared to theamount of thrombin generation prior to administration.

Yet an additional embodiment includes methods for inhibiting PAR-1,PAR-4 or both in a cell, by contacting the cell with an effective amountof a statin. This process can occur in vivo, in vitro or ex vivo, asfurther described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a new treatment for vascular disorders(e.g., heart disease and stroke). In particular, the present inventionis based on the unexpected discovery that certain receptors, known asG-coupled Protease Activating Receptor (PAR)-1 and PAR-4, are inhibitedby a statin, a lipid-lowering drug. Hence, the present inventionpertains to treating or preventing vascular disorders by administering astatin to inhibit PAR-1, PAR-4 or both.

Prior to the invention, it had been understood that statins can be usedto lower cholesterol levels by inhibiting HMG-CoA(3-hydroxy-3-methylglutaryl-coenzyme A) reductase, an enzyme thought tofacilitate the synthesis cholesterol. However, two surprising aspects ofthe invention exist. The first aspect is that statins, in addition toinhibiting HMG-CoA reductase, also inhibit PAR-1 and/or PAR-4 receptors.As such, patients who have cholesterol levels in normal ranges, but areat risk for heart disease or stroke (e.g., vascular events), caneffectively be treated with statins. Second, inhibiting PAR-1, PAR-4 orboth with statins actually reduce the incidence of vascular events.

The present invention pertains to methods for preventing or treating anindividual at risk for a (e.g., one or more) vascular event, disease ordisorder. The data described herein unexpectedly show that inhibitingPAR-1 and/or PAR-4 reduce the severity of or prevent vascular events.Prevention of a vascular event, condition, disease or disorder (e.g.,thrombotic event, condition, disease or disorder) refers to delaying orsuppressing the onset of the vascular disorder, or one or more of itssymptoms. To treat an individual at risk for a vascular disorder meansto alleviate, ameliorate or reduce the severity of one or more of itssymptoms.

Two phases of thrombotic events e.g., cardiovascular and/orcerebrovascular events, can exist: an ischemic stage and a necroticstage. A patient can suffer from ischemia in which a decrease of bloodflow can occur. This decrease in blood flow causes a decrease in tissueoxygenation. After prolonged ischemia, the tissue can undergo necrosiswhich is death of the tissue. Therefore, patients who are at risk for avascular event can exhibit elevated levels of ischemic markers and/ornecrosis markers.

An individual at risk for a vascular disorder refers to an individualwith a history of vascular disease, an individual experiencing at leastone symptom of the disorder, an individual having known risk factors(e.g., gender, weight) associated with or caused by the vasculardisorder, an individual undergoing a vascular procedure, or anindividual who has tested positive for a vascular condition using adiagnostic test (e.g., electrocardiogram, cardiac catheterization,stress test, ultrasound techniques, laboratory tests).

As described above, an embodiment of the invention includes treatingindividuals who are at risk for vascular events because they manifest atleast one symptom indicative of a vascular disorder/event. Symptoms thatare indicative of a coronary-related vascular event, for example,include chest pain, abnormal electrocardiograms, elevated levels ofischemic markers, necrosis markers, or thrombin/fibrin generationmarkers. Such markers include, but are not limited to, Creatine Kinasewith Muscle and/or Brain subunits (CKMB), D-Dimer, F1.2, thrombinanti-thrombin (TAT), soluble fibrin monomer (SFM), fibrin peptide A(FPA), myoglobin, thrombin precursor protein (TPP), platelet monocyteaggregate (PMA) and troponin. Individuals who are at risk also includethose having a history of a vascular event (e.g. disorder), includingCoronary Heart Disease (CHD), stroke, or Transient Ischemic Attacks(TIAs). A history of CHD can include, for example, a history of MI,coronary revascularization procedure, angina with ischemic changes, or apositive coronary angiogram (e.g., showing greater than about 50%stenosis of at least one major coronary artery).

The present invention also relates to methods for reducing theoccurrence or severity of a vascular disorder in a patient who is atrisk for such a disorder. Reducing the occurrence of a vascular disorder(e.g., a cardiovascular and/or cerebrovascular disorder) refers toreducing the probability that a patient will develop the disorder, ordelaying the onset of the disorder. Reducing the severity of a vasculardisorder refers to a reduction in the degree of at least one symptom ofthe disorder. The present invention embodies methods for preventing theonset of a vascular disorder in an individual having less than about a130 mg/dL LDL-C level, about a 200 mg/dL of Total-C or both byadministering a statin.

A vascular disorder is an event, disease or disorder that involves athrombosis (e.g., a thrombotic event) or a narrowing of a blood vessel.A vascular disorder/event occurs, for example, when a clot forms andlodges within a blood vessel. The blockage can fully block or partiallyblock the blood vessel causing a vascular disorder. Thrombin generationrefers to the activation, expression or up-regulation of thrombin, whichis involved in clot formation and an inductor of platelet activation.The amount of thrombin generation can be measured or assessed by certainmarkers known in the art. Examples of such thrombin generation markersinclude fibrinopeptide A, prothrombin fragments 1+2, andthrombin-antithrombin-III complexes.

Thrombin is a potent serine protease that plays a central role inhomeostasis following tissue injury by converting soluble plasmafibrinogen into an insoluble fibrin clot and by promoting plateletaggregation. In addition to these procoagulant effects, thrombin alsoinfluences a number of cellular responses that play important roles insubsequent inflammatory and tissue repair processes. Thrombin influencesthe recruitment and trafficking of inflammatory cells and is a potentmitogen for a number of cell types, including endothelial cells,fibroblasts, and smooth muscle cells. Thrombin also promotes theproduction and secretion of extracellular matrix proteins and influencesconnective tissue remodeling processes. There is increasing in vivoevidence that the pro-inflammatory and pro-fibrotic effects of thrombinplay an important role in both normal tissue and vascular repair, aswell as in a number of pathological conditions associated with acute orpersistent activation of the coagulation cascade, including restenosisand neointima formation following vascular injury, atherosclerosis,pulmonary fibrosis, and glomerulonephritis.

It has been determined that most of the cellular effects elicited bythrombin are mediated via a family of expressed PAR-1 receptors that areactivated by limited proteolytic cleavage of the N-terminalextracellular domain. The newly generated N-terminus acts as a tetheredligand and interacts intramolecularly with the body of the receptor toinitiate subsequent cell signaling events. When PAR-1 or PAR-4 isproteolytically cleaved, the new amino terminus functions as a tetheredligand to initiate signal transduction, and peptides derived from thenew amino terminus, thrombin receptor agonist peptides, function asagonists for the uncleaved receptor. As defined herein, thromboticevents include those involving thrombin formation. In one embodiment,the present invention relates to methods of preventing or treatingthrombin formation in individuals, as described herein, by administeringa statin.

Vascular events, diseases or disorders include cardiovascular diseases(e.g., coronary heart disease, myocardial infarction, angina or adisease in which a narrowing of a blood vessel occurs in at least onemajor artery), cerebrovascular diseases (e.g., stroke or transientischemic attacks), vascular procedures (e.g., thrombotic re-occlusionsubsequent to a coronary intervention procedure, heart or vascularsurgery) or any other thrombotic event (e.g., pulmonary embolism, deepvein thrombosis or peripheral vascular thrombosis). Vascular disordersalso include Syndrome X, which is a disease that is associated withunidentified chest pain. Vascular disorders include those in which atleast one major coronary artery exhibits greater than 50% stenosis.

Also, administering a statin can result in a reduction of recurrentvascular events (e.g., cardiovascular and/or cerebrovascular events).Use of the methods described herein results in a reduction of at leastabout 10% (e.g., 15%, 20%, 25%) in the number of recurrent heartattacks, cardiac deaths and/or strokes.

In one aspect, the present invention involves assessing the PAR-1 level,the PAR-4 level, or both to determine if the either or both levels areelevated. Individuals with elevated PAR-1 and/or the PAR-4 levels can beat risk for vascular events. Individuals can have other risk factors forvascular events, as further described herein. Administration of statinsinhibits PAR-1 and/or PAR-4, and levels of PAR-1 and/or PAR-4 arereduced, as compared to level(s) prior to administration of the statin.Controls and assessment of PAR-1 and/or PAR-4 are further describedherein. Statin administration prevents, reduces or treats the vasculardisorder.

A lipid profile is made up of a number of items including, but notlimited to, High Density Lipoprotein-Cholesterol (HDL-C), Low DensityLipoprotein-Cholesterol (LDL-C), triglycerides, and total Cholesterol(Total-C). Lipoproteins are complexes which contain both a lipid andprotein. Most of the lipids in plasma are present as lipoproteins andare transported as such. Lipoproteins are characterized by theirflotation constants (e.g., densities). Various classes of lipoproteinsexist and include HDLs and LDLs. LDLs are particularly rich incholesterol esters.

Levels of cholesterol that are considered normal can vary and depend onfactors such as the individual's health history, the number of riskfactors the individual has, etc. The National Cholesterol EducationProgram (NCEP) Guidelines state that a Total-C of greater than or equalto 200 mg/dL, and a LDL-C of greater than or equal to 130 mg/dL areconsidered borderline high. In one embodiment, the present inventionrelates to administering a (e.g., one or more) statin to an individualwith Total-C and/or LDL-C level(s) that are not considered to be high orborderline high (e.g., individuals with levels in normal ranges). Forexample, a normal range of Total-C is considered to be between about 60mg/dL and about 199 mg/dL, and LDL-C is between about 85 mg/dL and about129 mg/dL. As such, the present invention embodies administering astatin to an individual having a Total-C of less than about 200 mg/dL(e.g., between about 60 mg/dL and about 199 mg/dL) and/or a LDL-C ofless than about 130 mg/dL (e.g., between about 85 mg/dL and 129 mg/dL).Additionally, the present invention embodies administering a statin toan individual with triglyceride levels in normal ranges, e.g.,administering a statin to an individual with a triglyceride level ofless than 150 mg/dL (e.g., between about 30 mg/dL and about 149 mg/dL).

One embodiment of the invention includes administering a statin to anindividual having a Total-C and/or a LDL-C level in the upper end of thenormal range. For example, an individual having a LDL-C level in therange of about 100 mg/dL and about 129 mg/dL is considered to be in thenormal range, but not optimal. Similarly, an individual with a Total-Cin a range from about 170 mg/dL to about 199 mg/dL are also consideredto be normal, but not optimal. As such, individuals at risk for avascular event, as described herein, and have levels of LDL-C or Total-Cin the upper portion of the normal range can benefit from the invention.Hence, in one embodiment, the present invention relates to administeringa statin to an individual with a LDL-C level of between about 100 mg/dLand about 129 mg/dL, and/or a Total-C level of about 170 mg/dL and about199 mg/dL. Similarly, triglyceride levels that are on the upper end ofthe normal range include levels between about 100 mg/dL and about 149mg/dL. The invention therefore embodies administering a statin to anindividual at risk for a vascular event wherein the individual has atriglyceride level between about 100 mg/dL and about 149 mg/dL.

Lipoproteins levels and triglyceride levels are measured and assessedusing routine methods known in the art. Commercially available kits andassays can be used to evaluate the levels of Total-C, HDL-C, LDL-C andtriglycerides.

The present invention relates to methods of reducing or preventingvascular events by administering to an individual an effective amount ofa statin. The term, “statin” refers to a (e.g., one or more) compound ormetabolite thereof that inhibits HMG-CoA(3-hydroxy-3-methylglutaryl-coenzyme A) reductase, an enzyme thought tofacilitate the synthesis cholesterol. More particularly, statins inhibitthe conversion of HMG-CoA reductase to mevalonate, a precursor ofsterols, including cholesterol. Since the enzyme used to makecholesterol is blocked by the statin, then less cholesterol is made.Cholesterol and triglycerides circulate in the bloodstream as part oflipoprotein complexes. Hence, statins generally lower lipids in thisway. Additionally and more importantly, the data described hereindemonstrate that statins also block or inhibit PAR-1, PAR-4, or both,thereby reducing or preventing vascular events.

Examples of statins are atorvastatin (e.g., Atorvastatin Calcium,marketed under the trademark Lipitor® by Pfizer, Inc.), pravastatin(e.g., Pravastatin Calcium marketed under the trademark Pravachol® byBristol-Myers Squibb), fluvastatin (e.g., Fluvastatin Sodium marketedunder the trademark Lescol® by Novartis), cerivastatin (e.g.,Cerivastatin Sodium marketed under the trademark Baycol® by BayerCorporation), lovastatin (e.g., Lovastatin marketed under the trademarkMevacor® by Merck & Co., Inc.), simvastatin (e.g., Simvastatin marketedunder the trademark Zocor® by Merck & Co., Inc.), rosuvastatin (e.g.,Rosuvastatin Calcium marketed under the trademark Crestcor® byAstraZeneca, Inc.), pitavastatin (Nissan/Sankyo's nisvastatin (NK-104)or itavastatin), and metabolites thereof. The present inventionencompasses statins that are currently used, or those later discoveredor formulated. Statins include biologically active portions, groups orfragments of the statin that can inhibit PAR-1 or PAR-4.

Methods of making statins or isolating statins are known in the art.Statins can be made synthetically, or isolated from a bacteria (e.g.,Aspergillus terreus). Methods of making statins, their chemical formulasand their properties are described, for example, in U.S. Pat. Nos.3,983,140 (e.g., mevastatin); 4,231,938 (e.g., lovastatin); 4,346,227(e.g., pravastatin); 4,448,784 (e.g., simvastatin); 4,450,171 (e.g.,simvastatin); 5,354,772 (e.g., fluvastatin); 5,006,530 (e.g.,cerivastatin); 5,177,080 (e.g., cerivastatin); 4,681,893 (e.g.,atorvastatin); 5,273,995 (e.g., atorvastatin); 5,385,929 (e.g.,atorvastatin); 5,686,104 (e.g., atorvastatin); 5,260,440 (e.g.,rosuvastatin); and 5,011,930 (e.g., pitavastatin).

One aspect of the invention, as described herein, includes administeringa statin along with at least one other compound or composition that isused for treating the vascular condition (a “vascular treatingcompound”). For an individual with a cardiovascular disease, the statincan be administered together with aspirin, heparin, an ADP inhibitor orantagonist (e.g., thienopyridine, such as ticlopidine hydrochloride(marketed under the trademark “Ticlid®” from Roche Laboratories) orclopidogrel bisulfate (marketed under the trademark “Pravix®” fromBristol-Myers Squibb and Sanofi), GPIIb/IIIa inhibitors (marketed underthe trademark “ReoPro®” from Centocor, Inc.; “Integrilin” fromMillennium Pharmaceutical, Inc.; “Aggrastat” from GilfordPharmaceutical) or another statin. Individuals with cerebrovasculardiseases, for example, can receive a statin together with Ticlid®,Pravix® or aspirin.

The present invention involves inhibiting PAR-1, PAR-4, or both. Todetermine if PAR-1, PAR-4, or both are inhibited, one can assess orquantify these receptors. PAR-1 and/or PAR-4 can be measured todetermine whether an individual has elevated PAR-1 and/or PAR-4 levels,which would indicate that treatment with a statin is needed. In someembodiments, the present invention includes a specific step of assessingor measuring PAR-1, PAR-4, or both. Elevated PAR-1 and/or PAR-4 levelsdepend on the type of antibody used, e.g. greater than about 20 for theWEDE-15 antibody and about 40 for the SPAN-12 antibody (in log meanfluorescence).

The phrase “PAR receptors” refers to PAR-1 and/or PAR-4 receptors. “PARreceptor” refers to PAR-1 or PAR-4. In certain embodiments, theinvention refers to PAR-1 and/or PAR-4 receptors on the platelet.

A reduction in the PAR-1 and/or PAR-4 levels (e.g., platelet PAR-1and/or platelet PAR-4) refers to a decrease in or an absence of one orboth receptors, as compared to levels prior to administration or ascompared to a control, as further described herein. Levels of one orboth receptors can be decreased or reduced by at least about 10% (e.g.,20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), as compared to the leveljust prior to administration. Hence, one can measure the presence,absence or level of one or both of these receptors, and compare theresult against a control. For example, one can obtain a suitable sampleand compare the level of one or both of the receptors from previous timepoints (e.g., prior to administration of the statin or during the onsetof a vascular event, disease or disorder). PAR-1 and/or PAR-4 levelsdecrease after administration of the statin, as compared to the levelduring the onset of the vascular event. One can also measure PAR-1and/or PAR-4 levels in an individual prior to the onset of a vascularevent (e.g., in a resting state or during a check-up), to determine theindividual's baseline. Accordingly, administration of a statin after theonset of a vascular disease decreases the levels of PAR-1 and/or PAR-4levels, as compared to those levels during the onset of the vasculardisease, to reduce the level(s) so that they are closer to baseline(e.g., above, at or below baseline, but less than level(s) during theonset of the vascular disease).

The PAR-1 and/or PAR-4 levels assessed can also be compared to astandard or control obtained from normal individuals. In one example,PAR-1 and/or PAR-4 levels can be assessed in a population of healthyindividuals or individuals who have not had a vascular event, disease ordisorder. Such levels are referred to as a “negative control.”Conversely, PAR-1 and/or PAR-4 levels can also be obtained from a poolof individuals who are undergoing a vascular event, disease or disorder,e.g., a “positive control.” After administration of a statin, the levelof PAR-1, PAR-4 or both decreases; the level(s) get closer to the levelof the negative control, and farther from the positive control. ThePAR-1 and/or PAR-4 levels decrease as compared to the levels during theonset of the vascular event, disease or disorder. Hence, the methods ofthe present invention include reducing or inhibiting PAR-1 and/or PAR-4levels, with administration of a statin, wherein PAR-1 and/or PAR-4levels are reduced or decreased, as compared to those levels during theoccurrence of the vascular event, disease or disorder, or immediatelyprior to the administration of a statin.

In another embodiment, the present invention relates preventing theonset of a vascular event, disease or disorder. An effective amount ofat least one statin can be administered to prevent PAR-1 and/or PAR-4levels from increasing, or lessen PAR-1 and/or PAR-4 levels which wouldotherwise remain elevated without statin administration. For example, anindividual who is a risk for a vascular event, disease or condition cantake a statin on a daily basis (or every other day), to prevent PAR-1and/or PAR-4 levels from increasing as compared to a control orbaseline. Baseline levels of the activation state can be obtained priorto and/or during the course of administration of a statin. The PAR-1and/or PAR-4 levels can stay the same, or can even decrease. Similarly,PAR-1 and/or PAR-4 levels can be compared to a negative or positivecontrol, wherein upon administration of statin, the levels are closer tothe negative control, than the positive control. However measured, thePAR-1 and/or PAR-4 levels are prevented from increasing, therebypreventing the occurrence of a vascular event, disease or disorder.

The present invention also relates to reducing or inhibiting PAR-1and/or PAR-4 by contacting the platelets with a statin or metabolitethereof. This embodiment of the invention can be carried out in vivo, invitro or ex vivo. The method reduces PAR-1 and/or PAR-4 levels, ascompared to the level prior to contact of the statin or a metabolitethereof with the platelets.

Immunological Assessment of PAR-1 and PAR-4

Several suitable assays exist that measure the PAR receptors. Suitableassays encompass immunological methods, such as radioimmunoassay, flowcytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescenceassays, and assessment with a volumetric capillary cytometry system. Anymethod now known in the art or developed later can be used for measuringthe PAR receptors.

The assays utilize antibodies reactive with the PAR receptors, portionsthereof or functional fragments thereof. The terms polyclonal andmonoclonal refer to the degree of homogeneity of an antibodypreparation, and are not intended to be limited to particular methods ofproduction.

In several of the embodiments, immunological techniques detect PARreceptor levels by means of an anti-PAR antibody (i.e., one or moreantibodies). The phrase “anti-PAR receptor antibody” includes monoclonal(MoAbs) antibodies, polyclonal antibodies, and/or mixtures thereof. Forexample, these immunological techniques can utilize mixtures or acocktail of polyclonal and/or monoclonal antibodies.

Anti-PAR antibodies can be raised against an appropriate immunogen, suchas isolated and/or recombinant PAR receptors or portion thereof(including synthetic molecules, such as synthetic peptides). One canalso raise antibodies against a host cell which expresses recombinantPAR receptors. Additionally, cells expressing a recombinant PARreceptors, such as transfected cells, can be used as immunogens or in ascreen for antibody which binds receptor. Examples of PAR receptorantibodies are WEDE-15 MoAbs (PAR-1 and PAR-4) and SPAN-12 MoAbs (PAR-1and PAR-4).

Techniques known in the art can be employed to prepare an immunizingantigen and to produce polyclonal or monoclonal antibodies. The artcontains a variety of these methods (see e.g., Kohler et al., Nature,256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein etal., Nature 266: 550-552 (1977); Koprowski et al., U.S. Pat. No.4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.);Current Protocols In Molecular Biology, Vol. 2 (Supplement 27,Summer'94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York,N.Y.), Chapter 11, (1991)). Generally, fusing a suitable immortal ormyeloma cell line, such as SP2/0, with antibody producing cells canproduce a hybridoma. Animals immunized with the antigen of interest and,preferably, an adjuvant provides the antibody producing cell (cells fromthe spleen or lymph nodes). Selective culture conditions isolateantibody producing hybridoma cells while limiting dilution techniquesproduce them. One can use suitable assays such as ELISA to selectantibody producing cells with the desired specificity.

Other suitable methods can be employed to produce or isolate antibodiesof the requisite specificity. Examples of other methods includeselecting recombinant antibody from a library or relying uponimmunization of transgenic animals such as mice which are capable ofproducing a full repertoire of human antibodies (see e.g., Jakobovits etal., Proc. Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits etal., Nature, 362: 255-258 (1993); Lonberg et al., U.S. Pat. No.5,545,806; Surani et al., U.S. Pat. No. 5,545,807).

Immunological assays or techniques can be employed to determine thepresence, absence or level of PAR receptors in a biological sample. Indetermining the amounts of a membrane bound and/or soluble PARreceptors, an assay generally includes combining the sample to be testedwith an antibody having specificity for one or both of the PARreceptors, under conditions suitable for formation of a complex betweenantibody and the PAR receptor(s), and detecting or measuring (directlyor indirectly) the formation of a complex.

A sample can be obtained and prepared by a method suitable for theparticular sample (e.g., whole blood, platelet rich plasma), and selectthe assay format. For example, suitable methods for whole bloodcollection are venipuncture or obtaining blood from an in-dwellingarterial line. The container into which one deposits the blood cancontain an anti-coagulant such as CACD-A, heparin, or EDTA.

One or more PAR receptors can be measured in a sample with or withoutplatelets. To measure a soluble form of the PAR receptors, the plateletsare removed from the sample. A sample (e.g., blood) is collected, andplatelets are removed (partially or completely) from the sample, forexample, by preparation of serum or plasma (e.g., isolation of plateletpoor plasma). Samples are processed to remove platelets within a timesuitable to reduce artificial increases in soluble PAR receptors.Initiation of such processing within about one hour, and preferablyimmediately, is desirable. Samples can be further processed asappropriate (e.g., by dilution with assay buffer). Additionally, one canadd a reagent which stabilizes and prevents in vitro plateletactivation. Examples of these stabilizing reagents are apyrase and PGE₁.

Methods of combining sample and antibody, and methods of detectingcomplex formation are also selected to be compatible with the assayformat. Suitable labels can be detected directly, such as radioactive,fluorescent or chemiluminescent labels. They can also be indirectlydetected using labels such as enzyme labels and other antigenic orspecific binding partners like biotin. Examples of such labels includefluorescent labels such as fluorescein, rhodamine, CY5, APC,chemiluminescent labels such as luciferase, radioisotope labels such as³²P, ¹²⁵I, ¹³¹I, enzyme labels such as horseradish peroxidase, andalkaline phosphatase, β-galactosidase, biotin, avidin, spin labels andthe like. The detection of antibodies in a complex can also be doneimmunologically with a second antibody which is then detected.

Flow Cytometry:

One method for assessing PAR receptor levels is flow cytometry. Methodsof flow cytometry for measuring platelets or PAR receptors are known inthe art. (Shattil, Sanford J, et al. “Detection of Activated Plateletsin Whole Blood using Activation-Dependent Monoclonal Antibodies and FlowCytometry,” Blood, Vol. 70, No 1 (July), 1987: pp 307-315; Scharf,Rudiger E., et al., “Activation of Platelets in Blood PerfusingAngioplasty-damaged Coronary Arteries, Flow Cytometric Detection,”Arteriosclerosis and Thrombosis, Vol 12, No 12 (December), 1992: pp1475-1487.

For example, an assessment of one or more PAR receptors can be done. Asample comprising platelets is obtained from an individual. The sampleis contacted with an antibody having specificity for one or both PARreceptors under conditions suitable for formation of a complex betweenan antibody and the PAR receptor(s) expressed. A fluorescent label isused to detect the complex formation, either directly or indirectly. Thein vivo affect of a statin is assessed by obtaining samples atparticular time points (e.g., a baseline, during a vascular event, afteradministration of a statin, etc.), as described herein, and measuringthe presence, absence, or level one or more PAR receptors.

For ex vivo assessment of the statins effect on a PAR receptor, a levelof a PAR receptor can be assessed by flow cytometry by first obtaining asample that comprises platelets and then contacting the sample with aplatelet activation agonist, such as phorbol myristate acetate (PMA),ADP (adenosine diphosphate), thrombin, collagen, and/or TRAP (thrombinreceptor activating peptide), under conditions suitable for activationof platelets in the sample. The sample is in contact with the agonistpreferably for a period of time effective to maximally activate theplatelets. The sample is then subjected to a statin at particularconcentrations. Then one contacts the samples with a composition thatcomprises an anti-PAR antibody e.g., having a fluorescent label,preferably in an amount in excess of that required to bind the PARreceptor expressed on the platelets, under conditions suitable for theformation of labeled complexes between the anti-PAR antibody andactivated platelets. Then the formation of the complex in the sample isassessed (e.g., detected or measured).

The sample can be divided to form controls. For example, a portion ofthe sample can be maximally activated and not contacted with a statin.Also, one can obtain a portion of the sample and not expose it to aplatelet activation agonist, nor the statin to determine a baselinelevel of the PAR receptors. See Exemplification for detailed descriptionof the flow cytometry methods. This ex vivo method is not limited toflow cytometry, but can also be used in other methods for assessing PARreceptor levels. Receptor expression is presented as Log meanfluorescence intensity.

Radioimmunoassay:

In addition to using flow cytometry to measure a PAR receptor, aradioimmunoassay can be employed. A PAR receptor can be assessed by aradioimmunoassay by first obtaining a suitable sample to be tested. Thesample is contacted with an anti-PAR antibody (e.g., an anti-PARantibody comprising a radioactive label, or an anti-PAR antibodycomprising a binding site for a second antibody that has a radioactivelabel) preferably in an amount in excess of that required to bind thePAR receptors expressed on the platelets, and under conditions suitablefor the formation of labeled complexes between the anti-PAR antibody andactivated platelets. The formation of the complex in the samples isdetermined by detecting or measuring the radioactivity in the sample.

Enzyme-Linked Immunosorbent Assays (ELISA):

Detection of a PAR receptor in a suitable sample can also occur byemploying ELISA methods. To determine a measurement of a PAR receptorusing an ELISA assay in a suitable sample, one contacts the sample withan anti-PAR antibody, and then measures the formation of a complexbetween the anti-PAR antibody and the PAR receptor(s) in the sample. ThePAR receptor(s) can be measured by direct, indirect, sandwich orcompetitive ELISA formats. An antibody can be conjugated with labelssuch as biotin and HRP-streptavidin.

A solid support, such as a microtiter plate, dipstick, bead, or othersuitable support, can be coated directly or indirectly with an anti-PARantibody. For example, an anti-PAR antibody can coat a microtiter well,or a biotinylated anti-PAR MoAbs can be added to a streptavidin coatedsupport. A variety of immobilizing or coating methods as well as anumber of solid supports can be used, and can be selected according tothe desired format.

In one embodiment, the sample or PAR receptor standard is combined withthe solid support simultaneously with the detector antibody. Optionally,this composition can be combined with one or more reagents by whichdetection is monitored. For example, the sample such as PPP can becombined with the solid support simultaneously with (a) HRP-conjugatedanti-PAR MoAbs, or (b) a biotinylated anti-PAR MoAbs andHRP-streptavidin.

A known amount of the PAR receptor standard can be prepared andprocessed as described above for a suitable sample. This PAR receptorstandard assists in quantifying the amount of PAR receptors detected bycomparing the level of PAR receptors in the sample relative to that inthe standard.

A physician, technician, apparatus or a qualified person can compare theamount of detected complex with a suitable control to determine if thelevels are decreased. For example, the level of a PAR receptor followinga vascular intervention procedure can be compared with a basal level forthe individual, such as a level determined prior to or at the time ofthe procedure, or with levels in normal individuals or suitablecontrols, as described herein.

A variety of methods can determine the amount of PAR receptors incomplexes. For example, when HRP is used as a label, a suitablesubstrate such as OPD can be added to produce color intensity directlyproportional to the bound anti-PAR MoAbs (assessed e.g., by opticaldensity), and therefore to the PAR receptors in the sample. One cancompare the results to a suitable control such as a standard, levels ofPAR receptors in normal individuals, and baseline levels of PARreceptors in a sample from the same donor. For example, the assay can beperformed using a known amount of a PAR receptor standard in lieu of asample, and a standard curve established. One can relatively compareknown amounts of the PAR receptor standard to the amount of complexformed or detected.

PAR-1 and/or PAR-4 levels can be assessed using methods that arecurrently known in the art, as well as those that are later discovered.

Modes and Manner of Administration, Dosages

The statins used in the present invention can be administered with orwithout a carrier. The terms “pharmaceutically acceptable carrier” or a“carrier” refer to any generally acceptable excipient or drug deliverycomposition that is relatively inert and non-toxic. Exemplary carriersinclude sterile water, salt solutions (such as Ringer's solution),alcohols, gelatin, talc, viscous paraffin, fatty acid esters,hydroxymethylcellulose, polyvinyl pyrolidone, calcium carbonate,carbohydrates (such as lactose, sucrose, dextrose, mannose, albumin,starch, cellulose, silica gel, polyethylene glycol (PEG), dried skimmilk, rice flour, magnesium stearate, and the like. Suitableformulations and additional carriers are described in Remington'sPharmaceutical Sciences, (17^(th) Ed., Mack Pub. Co., Easton, Pa.). Suchpreparations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,preservatives and/or aromatic substances and the like which do notdeleteriously react with the active compounds. Typical preservatives caninclude potassium sorbate, sodium metabisulfite, methyl paraben, propylparaben, thimerosal, etc. The compositions can also be combined wheredesired with other active substances, e.g., enzyme inhibitors, to reducemetabolic degradation. A carrier (e.g., a pharmaceutically acceptablecarrier) is preferred, but not necessary to administer the compound.

The statin can be a liquid solution, suspension, emulsion, tablet, pill,capsule, sustained release formulation, or powder. The method ofadministration can dictate how the composition will be formulated. Forexample, the composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc.

The statins used in the invention can be administered intravenously,parenterally, intramuscular, subcutaneously, orally, nasally, topically,by inhalation, by implant, by injection, or by suppository. Thecomposition can be administered in a single dose or in more than onedose over a period of time to confer the desired effect. In oneembodiment, atorvastatin, pravastatin, fluvastatin, cerivastatin,lovastatin, simvastatin, rosuvastatin, or pitavastatin, can beadministered orally in an amount between about 2 mg-100 mg/daily (e.g.,10, 20, 40 or 80 mg/daily).

The actual effective amounts of compound or drug can vary according tothe specific composition being utilized, the mode of administration andthe age, weight and condition of the patient. For example, as usedherein, an effective amount of the drug is an amount which reduces theplatelet activation state. Dosages for a particular individual patientcan be determined by one of ordinary skill in the art using conventionalconsiderations, (e.g. by means of an appropriate, conventionalpharmacological protocol).

For enteral or mucosal application (including via oral and nasalmucosa), particularly suitable are tablets, liquids, drops,suppositories or capsules. A syrup, elixir or the like can be usedwherein a sweetened vehicle is employed. Liposomes, microspheres, andmicrocapsules are available and can be used.

Pulmonary administration can be accomplished, for example, using any ofvarious delivery devices known in the art such as an inhaler. See. e.g.,S. P. Newman (1984) in Aerosols and the Lung, Clarke and Davis (eds.),Butterworths, London, England, pp. 197-224; PCT Publication No. WO92/16192; PCT Publication No. WO 91/08760.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Inparticular, carriers for parenteral administration include aqueoussolutions of dextrose, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylene-polyoxypropylene blockpolymers, and the like. Ampules are convenient unit dosages.

The administration of the statin and the vascular treating compound canoccur simultaneously or sequentially in time. The vascular treatingcompound can be administered before, after or at the same time as thestatin. Thus, the term “co-administration” is used herein to mean thatthe statin and the vascular treating compound will be administered attimes to achieve a reduction or treatment of the vascular event ordisease, and inhibition of PAR-1 or PAR-4. The methods of the presentinvention are not limited to the sequence in which the statin andvascular treating compound are administered, so long as the vasculartreating compound is administered close enough in time to produce thedesired effect of reducing the platelet activation state.

A description of preferred embodiments of the invention follows.

EXEMPLIFICATION Statins Inhibit PAR-1 and PAR-4 Methods: Patients:

The study was designed as a prospective, single blinded, placebocontrolled trial of 6 major statins currently available in the US. Thestudy was approved by local institutional review boards. Writteninformed consent was obtained from all patients, who were informed ofthe strict compliance rules, and compensated for outpatient visits, andblood draws. Patients aged >21 years were eligible if they had adocumented evidence of coronary artery disease (proved by coronaryangiography).

Patients were excluded for a history of bleeding diathesis, drug oralcohol abuse, prothrombin time greater than 1.5 times control, plateletcount <100,000/mm³, hematocrit <25%, or creatinine >4.0 mg/dl, surgeryor angioplasty for symptomatic stenosis performed within 3 months orplanned for the future, known allergy to aspirin, clopidogrel, orstatins, history of gastrointestinal or other bleeding, history ofdrug-induced disorders, trauma or surgery within the last 3 months, anysurgery planned for the next 3 months, cancer, rheumatic diseases, orseizures. Patients participating in other investigational drug trialswithin one month of completion were also excluded. No patients hadpreviously received intravenous platelet glycoprotein IIb/IIIainhibitors for the last 6 months.

Samples

Blood samples were obtained with a 19-gauge needle by directvenipuncture and drawn into two 7-ml vacutainer tubes at roomtemperature containing 3.8% trisodium citrate. The vacutainer tube wasfilled to capacity and gently inverted 3 to 5 times to ensure completemixing of the anticoagulant. The first 4-5 ml of blood were used forlipid profile analysis, or discharged. All samples were labeled withcoded number and analyzed by blinded technicians. Platelet studies wereperformed at baseline as well as at Week 4, and Week 6.

Measuring PAR-1/PAR-4 Receptors by Whole Blood Flow Cytometry

The blood-citrate mixture (50 μl) was diluted with 450 μl Tris bufferedsaline (TBS) (10 mmol/l Tris, 0.15 mol/l sodium chloride) and mixed byinverting an Eppendorf tube gently two times. The appropriate antibodywas then added (5 μl) and incubated at 37° C. for 30 minutes, afterincubation, 400 μl of 2% buffered paraformaldehyde was added forfixation. The samples were analyzed on the FACScan flow cytometer(Becton Dickinson, San Diego, Calif.) calibrated to measure fluorescentlight scatter. All parameters were collected using four-decadelogarithmic amplification. The data were collected in list mode filesand then analyzed. The surface expression of platelet receptors wasdetermined by flow cytometry using the following monoclonal antibodies:cleaved (WEDE15), and intact (SPAN12) platelet thrombin PAR-1/PAR-4receptors (Beckman Coulter, Brea, Calif.), and they were expressed aslog mean fluorescence intensity.

Statistical Analysis

The significance of differences between treatments arms was calculatedby c2 and Fisher's exact tests for discrete variables, and Wilcoxonrank-sum test for continuous variables. The significance of differencesbetween individual flow cytometric histograms was calculated using theSmirnov-Kolgomorov test incorporated in the CELLQuest' (San Diego,Calif.) software. Statistical analyses were performed using SPSS/E11.5(SPSS, Inc., Chicago, Ill.). To control for any baseline differencesanalysis of variance was used. All p values are 2 sided.

Results Patients:

Seventy patients were screened in, assigned, and completed the six weeksstudy. There were no deaths, serious adverse events, orhospitalizations. Table 1 shows baseline pretreatment distribution ofdemographics, risk factors, clinical characteristics, and concomitantmedications in 6 statin-, and one control arms. There were nosignificant differences between the groups, and concomitant medicationswere used fairly even.

TABLE 1 Baseline Clinical Characteristics of Patients with CoronaryArtery Disease No statins Atorvastatin Rosuvastatin PravastatinSimvastatin Fluvastatin Lovastatin Variable (n = 10) (n = 10) (n = 10)(n = 10) (n = 10) (n = 10) (n = 10) Age, yrs ± SD 61.8 ± 7.0  64.2 ±10.1 62.5 ± 7.7 65.5 ± 6.4 58.3 ± 9.1  57.9 ± 11.0 65.2 ± 6.8 Gender, n(% male) 8 (60%) 8 (80%) 6 (60%) 6 (60%) 8 (80%) 7 (70%) 6 (60%) Race, n(%) Caucasian 7 (70%) 9 (90%) 6 (60%) 8 (80%) 8 (80%) 8 (80%) 7 (70%)African-American 3 (30%) — 4 (40%) 2 (20%) 1 (10%) 1 (10%) — Asians — 1(10%) — — 1 (10%) 1 (10%) 3 (30%) Primary Diagnosis, n (%) Stable angina9 (90%) 8 (80%) 7 (70%) 10 (100%) 8 (80%) 9 (90%) 8 (80%) Unstableangina 1 (10%) 1 (10%) 1 (10%) — — — 2 (20%) Other — 1 (10%) 2 (20%) — 2(20%) 1 (10%) — Risk factors Total cholesterol, ± SD 244 ± 52 275 ± 33301 ± 59 282 ± 39 197 ± 25 261 ± 30 274 ± 42 Triglycerides, ± SD 155 ±61 168 ± 57 174 ± 52 160 ± 41 148 ± 55 180 ± 58 161 ± 50 HDL-C, ± SD  37± 11  40 ± 10 36 ± 7 41 ± 9 38 ± 8 35 ± 6 37 ± 6 LDL-C, ± SD 127 ± 48130 ± 39 135 ± 34 142 ± 30 140 ± 43 129 ± 30 135 ± 38 Tobacco use, n (%)5 (50%) 4 (40%) 4 (40%) 6 (60%) 3 (30%) 5 (50%) 4 (40%) Hypertension, n(%) 8 (80%) 9 (90%) 9 (90%) 8 (80%) 7 (70%) 7 (70%) 7 (70%) Diabetes, n(%) 4 (40%) 4 (40%) 6 (60%) 6 (60%) 5 (50%) 3 (30%) 5 (50%) MedicalHistory, n (%) Previous MI — — — 1 (10%) 2 (20%) — — Heart Failure 1(10%) — — 1 (10%) 1 (10%) 1 (10%) 2 (20%) Peripheral vascular disease 3(30%) 3 (30%) 2 (20%) 2 (20%) 3 (30%) 2 (20%) 2 (20%) Heart surgery 1(10%) — — 2 (20%) 1 (10%) — — Medications, n (%) Beta-blockers 6 (60%) 6(60%) 4 (40%) 4 (40%) 7 (70%) 7 (70%) 5 (50%) ACE inhibitors 3 (30%) 2(20%) 3 (30%) 2 (20%) 1 (10%) 3 (30%) 3 (30%) Ca-channel blockers 1(10%) 1 (10%) 2 (20%) 3 (30%) 2 (20%) 2 (20%) — AT-receptor antagonists4 (40%) 4 (40%) 5 (50%) 3 (30%) 5 (50%) 4 (40%) 5 (50%) Antidepressants3 (30%) 2 (20%) 3 (30%) 4 (40%) 4 (40%) 2 (20%) 1 (10%) Aspirin 8 (80%)10 (100%) 10 (100%) 9 (90%) 10 (100%) 10 (100%) 9 (90%) Clopidogrel 3(30%) 4 (40%) 3 (30%) 3 (30%) 5 (50%) 4 (40%) 2 (20%) Warfarin 1 (10%) —— 1 (10%) 1 (10%) — —

Statin Doses:

Patients were assigned to 6 major statins, or serve as a control groupwith no statin therapy. The brand and dose of statin was at physiciandiscretion. Patients were assigned in each study arm until the cell isfilled. Table 2 exhibit the frequency of different statin doses used inthe index study

PAR-1/PAR-4 Expression:

PAR-1 is a member of a novel gene family of G-protein couples receptors.PAR-1 is a single polypeptide of 66 kDa with a thrombin cleavage sitelocated near the extracellular N terminus. This receptor is expressed byhuman platelets and is responsible for attracting alpha-thrombin to theplatelet surface. The SPAN12 monoclonal antibody used in this studyspecifically reacts with PAR-1. It was found that a consistent andsignificant reduction of PAR-1 platelet expression in the statin treatedpatients independently of the brand of statin used.

More specifically, the data on PAR-1/PAR-4 thrombin receptor expressionin all groups are presented in Table 3. At baseline, there was nodifference in receptor expression. However, 4 weeks of treatment withstatins resulted in a significant inhibition of the activated epitope ofPAR-1/PAR-4 platelet expression as measured by WEDE-15 monoclonalantibody. There was a slight rebound at week 6, but still significantinhibition was observed. The pattern of inhibition of non-activatedintact epitope (measured with SPAN-12 antibody) was delayed. For mostgroups it takes six weeks (with the exception of simvastatin andpravastatin) of therapy to develop the inhibition of the intactreceptor.

TABLE 2 Doses of statins used in the platelet PAR-1/PAR-4 studies.Statin Atorvastatin Rosuvastatin Pravastatin Simvastatin FluvastatinLovastatin Daily Dose (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n =10)  5 mg N/A 1 (10%) N/A 0 N/A N/A 10 mg 2 (20%) 7 (70%) 2 (20%) 2(20%) N/A 5 (50%) 20 mg 4 (40%) 2 (20%) 7 (70%) 3 (30%) 2 (20%) 4 (40%)40 mg 4 (40%) 0 1 (10%) 4 (40%) 6 (60%) 1 (10%) 80 mg 0 N/A N/A 1 (10%)2 (20%) N/A

TABLE 3 Effects of Statins on the Platelet PAR-1/PAR-4 receptorsPatients with Coronary Artery Disease No statins AtorvastatinRosuvastatin Pravastatin Simvastatin Fluvastatin Lovastatin Variable (n= 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) WEDE15-MoAbs(a cleaved epitope of (PAR-1/PAR-4 receptors) Baseline 24.4 ± 5.4 27.5 ±4.9 30.1 ± 7.2 28.2 ± 4.4 29.0 ± 7.3 27.1 ± 5.9 26.8 ± 4.9 Week 4 27.1 ±6.1 16.1 ± 5.8* 17.4 ± 5.5* 16.9 ± 4.8* 16.6 ± 5.5* 18.0 ± 5.8* 18.4 ±5.0* Week 6 25.0 ± 8.3 18.0 ± 7.4* 22.6 ± 7.2* 20.1 ± 6.6* 23.4 ± 8.120.7 ± 4.1* 23.0 ± 6.3 SPAN-12-MoAbs (nonactive, intact epitope ofPAR-1/PAR-4 receptors) Baseline 43.6 ± 5.8 46.1 ± 8.3 42.2 ± 9.4 50.3 ±9.1 45.4 ± 7.0 42.8 ± 8.2 40.6 ± 6.9 Week 4 40.4 ± 6.0 39.5 ± 5.7 33.6 ±5.9 29.0 ± 6.7* 26.5 ± 5.8* 37.1 ± 7.3 40.0 ± 8.1 Week 6 50.7 ± 5.2 30.8± 4.0* 25.1 ± 7.0* 32.6 ± 8.4* 24.9 ± 6.2* 29.9 ± 6.4* 31.1 ± 5.5*Receptor expression is presented as Log mean fluorescence intensity; *p< 0.05

The relevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details can bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a vascular disorder associated with increasedPAR-1, PAR-4 or both in an individual, wherein the method comprises: a)selecting an individual diagnosed with a vascular disorder characterizedby having an elevated G-coupled Protease Activating Receptor (PAR)-1level, an elevated PAR-4 level, or both; b) administering to saidindividual an amount of a statin, wherein the statin is administered inan amount between 5 mg and 250 mg to thereby inhibit PAR-1, PAR-4 orboth; and c) detecting in said individual the level of PAR-1, PAR-4 orboth and comparing said level to step a); wherein the statin reduces thePAR-1 level, the PAR-4 level or both, as compared to the level prior tostep b), to thereby treat the vascular disorder.
 2. The method of claim1, further comprising selecting an individual who has aTotal-Cholesterol (Total-C) or Low Density Lipoprotein Cholesterol(LDL-C) level in a normal range.
 3. The method of claim 1, wherein theindividual has a Total-C level of less than about 200 mg/dL.
 4. Themethod of claim 1, wherein the individual has a LDL-C level of less thanabout 130 mg/dL.
 5. The method of claim 1, wherein the vascular disorderis selected from the group consisting of myocardial infarction, angina,stroke, pulmonary embolism, transient ischemic attack, deep veinthrombosis, thrombotic re-occlusion subsequent to a coronaryintervention procedure, heart surgery or vascular surgery, peripheralvascular thrombosis, Syndrome X, heart failure and a disorder in which anarrowing of at least one coronary artery occurs.
 6. The method of claim1, wherein the statin is selected from the group consisting ofatorvastatin, an atorvastatin metabolite, pravastatin, a pravastatinmetabolite, fluvastatin, a fluvastatin metabolite, cerivastatin, acerivastatin metabolite, lovastatin, a lovastatin metabolite,simvastatin, a simvastatin metabolite, rosuvastatin, rosuvastatinmetabolite, pitavastatin and a pitavastatin metabolite.
 7. (canceled) 8.The method of claim 1, wherein PAR-1 or PAR-4 are found on platelets. 9.A method for treating a vascular disorder associated with increasedPAR-1, PAR-4 or both in an individual, wherein the method comprises: a)assessing a level of PAR-1, PAR-4 or both in the individual, andcomparing said levels to a control, wherein an elevated level of PAR-1,PAR-4 or both is determined; and b) administering to said individual anamount of a statin, wherein the statin is administered in an amountbetween 5 mg and 250 mg to thereby inhibit PAR-1, PAR-4 or both; whereinthe statin reduces the PAR-1 level, PAR-4 level or both, as compared tothe level prior to step b) to thereby treat the vascular disorder. 10.The method of claim 9, further comprising reducing levels of PAR-1,PAR-4 or both by at least 10%, as compared to the elevated levels ofstep a).
 11. The method of claim 10, further comprising selecting anindividual who has a Total-C or LDL-C level in a normal range.
 12. Themethod of claim 9, wherein the vascular disorder is selected from thegroup consisting of myocardial infarction, angina, stroke, pulmonaryembolism, transient ischemic attack, deep vein thrombosis, thromboticre-occlusion subsequent to a coronary intervention procedure, heartsurgery or vascular surgery, peripheral vascular thrombosis, Syndrome X,heart failure and a disorder in which a narrowing of at least onecoronary artery occurs.
 13. The method of claim 9, further comprisingadministering a vascular treating compound.
 14. The method of claim 9,wherein the statin is selected from the group consisting ofatorvastatin, an atorvastatin metabolite, pravastatin, a pravastatinmetabolite, fluvastatin, a fluvastatin metabolite, cerivastatin, acerivastatin metabolite, lovastatin, a lovastatin metabolite,simvastatin, a simvastatin metabolite, rosuvastatin, rosuvastatinmetabolite, pitavastatin and a pitavastatin metabolite.
 15. A method oftreating an individual with a vascular disorder, wherein the methodcomprises: a) selecting an individual diagnosed with a vascular disordercharacterized by having an elevated PAR-1 level, an elevated PAR-4level, or both; and has a Total-C level, a LDL-C level, or both in anormal range; and b) administering to said individual an amount of astatin, wherein the statin is administered in an amount between 5 mg and250 mg to thereby inhibit PAR-1, PAR-4 or both; wherein PAR-1, PAR-4 orboth are inhibited to thereby treat the vascular disorder.
 16. Themethod of claim 15, wherein the individual has a Total-C level of lessthan about 200 mg/dL.
 17. The method of claim 15, wherein the individualhas a LDL-C level of less than about 130 mg/dL.
 18. A method of reducingthrombin generation in an individual, wherein the method comprises: a)selecting an individual with a vascular disorder characterized by havingan elevated PAR-1 level, an elevated PAR-4 level, or both and has aTotal-C level of less than about 200 mg/dL, a LDL-C level of less thanabout 130 md/dL, or both; and b) administering to said individual anamount of a statin, wherein the statin is administered in an amountbetween 5 mg and 250 mg to thereby inhibit PAR-1, PAR-4 or both; whereinthrombin generation is reduced, as compared to the thrombin generationprior to step b).
 19. The method of claim 18, wherein the reduction ofthrombin generation thereby treats or prevents a vascular disorderselected from the group consisting of myocardial infarction, angina,stroke, pulmonary embolism, transient ischemic attack, deep veinthrombosis, thrombotic re-occlusion subsequent to a coronaryintervention procedure, heart surgery or vascular surgery, peripheralvascular thrombosis, Syndrome X, heart failure and a disorder in which anarrowing of at least one coronary artery occurs.
 20. The method ofclaim 18, wherein inhibiting PAR-1, PAR-4 or both comprises reducinglevels of PAR-1, PAR-4 or both by at least 10%, as compared to levelsprior to step b).
 21. The method of claim 18, wherein the statin isselected from the group consisting of atorvastatin, an atorvastatinmetabolite, pravastatin, a pravastatin metabolite, fluvastatin, afluvastatin metabolite, cerivastatin, a cerivastatin metabolite,lovastatin, a lovastatin metabolite, simvastatin, a simvastatinmetabolite, rosuvastatin, rosuvastatin metabolite, pitavastatin and apitavastatin metabolite.
 22. A method of preventing a vascular disorderassociated with increased PAR-1, PAR-4, or both in an individual,wherein the method comprises: a) selecting an individual at risk for thevascular disorder characterized by having an elevated PAR-1 level, anelevated PAR-4 level, or both; and b) administering to said individualan amount of a statin, wherein the statin is administered in an amountbetween 5 mg and 250 mg to thereby inhibit PAR-1, PAR04 or both whereinthe statin reduces the PAR-1 level, the PAR-4 level or both, as comparedto the level prior to step b), to thereby prevent the vascular disorder.23. A method of preventing a vascular disorder associated with increasedPAR-1, PAR-4 in an individual, wherein the method comprises: a)assessing a level of PAR-1, PAR-4 or both in the individual, andcomparing said levels to a control, wherein an elevated level of PAR-1,PAR-4 or both is determined; and b) administering to said individual anamount of a statin, wherein the statin is administered in an amountbetween 5 mg and 250 mg; wherein the statin reduces the PAR-1 level, thePAR-4 level, or both as compared to the level prior to step b), tothereby prevent the vascular disorder.
 24. A method of preventing avascular disorder associated with increased PAR-1, PAR-4 or both in anindividual, wherein the method comprises: a) selecting an individual atrisk for the vascular disorder characterized by having an elevatedG-coupled Protease Activating Receptor (PAR)-1 level, an elevated PAR-4level, or both, wherein the individual has a Total-C level, a LDL-Clevel, or both in a normal range; and b) administering to saidindividual an amount of a statin, wherein the statin is administered inan amount between 5 mg and 250 mg to thereby inhibit PAR-1, PAR-4 orboth; wherein the statin reduces the PAR-1 level, PAR-4 level or both ascompared to the level prior to step b), to thereby prevent the vasculardisorder.
 25. A method of preventing or reducing thrombin formation inan individual, wherein the method comprises: a) selecting an individualhaving a thrombin formation or an individual who is at risk for thethrombin formation; and b) administering to the individual an effectiveamount of a statin; wherein PAR-1, PAR-4 or both are inhibited.
 26. Themethod of claim 25, wherein the individual has a Total-C level, LDL-Clevel, or both in a normal range.
 27. A method of inhibiting PAR-1,PAR-4 or both in a cell, wherein said the method comprises contactingthe cell with an effective amount of a statin to thereby inhibit PAR-1,PAR-4 or both.
 28. The method of claim 27, wherein the cell is contactedex vivo.